Hemostasis is maintained by a complex interplay of a variety of enzymes. Clotting factors interact in a "cascade" of activation steps which eventually leads to the formation of a fibrin clot. Subsequent degradation of the fibrin clot is accomplished by the fibrinolytic system, which involves the serine protease plasmin, a proteolytic enzyme which breaks down fibrin. Plasmin is a broad spectrum protease which also cleaves certain coagulation factors, thereby inactivating them. Production of plasmin from its inactive precursor, plasminogen, is mediated by tissue plasminogen activator (t-PA) , a fibrin-specific serine protease which is believed to be the physiological vascular activator of plasminogen. Urokinase-type plasminogen activator (u-PA) is another member of the class of plasminogen activators characterized as serine proteases. t-PA and u-PA are functionally and immunologically distinguishable. If the normal hemostatic system becomes upset, clots may form at inappropriate times and places, leading to myocardial infarction, deep vein thrombosis, pulmonary embolism and stroke. Tissue damage resulting from these conditions may result in death or serious disability.
t-PA normally circulates as a single polypeptide chain of M.sub.r =72,000 daltons, which is converted to a two-chain form by cleavage of a peptide bond between amino acids 275 (Arg) and 276 (Ile). The heavy chain of t-PA (two variants of M.sub.r 40,000 and 37,000) is derived from the amino-terminus, while the light chain (M.sub.r 33,000) is derived from the carboxy-terminal end of the t-PA molecule. This cleavage is catalyzed by trypsin or plasmin, and is accompanied by an increase in activity, as measured using synthetic substrates, and by an increase in fibrinolytic activity. Single-chain t-PA becomes active upon binding to fibrin, probably due to a conformational change in the activator induced by binding to fibrin. Cleavage to the two-chain form may be associated with rapid clearance of t-PA from the bloodstream, but conflicting reports on this have been published (see Wallen et al., Eur. J. Biochem. 132:681-686, 1983), and the clearance mechanism is poorly understood.
A two-dimensional model of the potential precursor t-PA protein has been established (Ny et al., Proc. Natl. Acad. Sci. USA 81:5355-5359, 1984). From this model, it was determined that the heavy chain contains two triple disulfide structures known as "kringles." Similar kringle structures also occur in prothrombin, plasminogen and urokinase, and are believed to be important for binding to fibrin (Ny et al., ibid.). The second kringle (K2) of t-PA is believed to have a higher affinity for fibrin than the first kringle (K1) (Ichinose, Takio and Fujikawa, J. Clin. Invest. 78:163-169, 1986; Verheijen et al., EMBO J. 5:3525-3530, 986).
In addition, the heavy chain of t-PA contains a "growth factor" domain, a triple disulfide-bonded structure which has homology to epidermal growth factor and to similar domains in protein C, factor VII, factor IX and factor X. The growth factor domain of native t-PA encompasses approximately amino acids 48-90.
The heavy chain of t-PA also contains a "finger" domain that is homologous to the finger domains of fibro-nectin. Fibronectin exhibits a variety of biological activities, including fibrin binding; its fibrin-binding activity has been correlated to four or five of its nine finger domains.
The light chain of t-PA contains the active site for serine protease activity (the serine protease domain), which is highly homologous to the active sites of other serine proteases.
The precursor form of t-PA additionally comprises a pre-region followed downstream by a pro-region, which are collectively referred to as the "pre-pro" region. The pre-region contains a signal peptide which is important for secretion of t-PA by vascular endothelial cells (Ny et al., ibid.). The pre sequence is believed responsible for secretion of t-PA into the lumen of the endoplasmic reticulum, a necessary step in extracellular secretion. The pro sequence is believed to be cleaved from the t-PA molecule following transport from the endoplasmic reticulum to the Golgi apparatus.
The use of t-PA for fibrinolysis in animal and human subjects has highlighted several shortcomings of the native molecule. The half-life of t-PA in vivo has been shown to be as short as three minutes in humans (Nilsson et al., Scand. J. Haematol. 33:49-53, 1984). Injected t-PA is rapidly cleared by the liver, and, within 30 minutes, most of the injected material is metabolized to low molecular weight forms. This short half-life may result in a need for high therapeutic dosages. Typically, native t-PA is administered at a dose of 30 to 150 mg per patient, and the low solubility of the protein necessitates prolonged infusion. Fuchs et al. (Blood 65:539-544, 1985) concluded-that infused t-PA is cleared by the liver in a process independent of the proteolytic site and that infused t-PA will not accumulate in the body; that is, the clearance mechanism cannot be saturated. Furthermore, doses of t-PA sufficient to lyse coronary thrombi are far larger than normal physiological levels, and may cause activation of plasminogen throughout the body, leading to systemic degradation of fibrinogen (Sherry, ibid.), which results in dangerous bleeding episodes.
Various workers have modified t-PA in attempts to enhance its clinical suitability. Rosa and Rosa (International Patent Application WO 86/01538) modified the Lys at position 277 of t-PA to stabilize the single-chain form of t-PA. Ile (277) t-PA produced in E. coli was found to be less active as a single-chain molecule, as compared to native t-PA. Wallen et al. (ibid.) postulated that the lysine residue at position 277 may be responsible for the proteolytic activity of single-chain t-PA. Heyneker and Vehar (published British Patent Application 2,173,804) disclose amino acid substitutions around the cleavage site of t-PA. A variant t-PA comprising Glu at position 275 was shown to have greater specific activity as compared to native t-PA. This variant t-PA also formed fewer complexes with t-PA inhibitor. The single-chain form was also shown to have greater affinity for fibrin than the two-chain form. Robinson (WO 84/01786) used enzymatic means to remove carbohydrate side chains from t-PA to increase plasma half-life. Lau et al. (Bio/Technology 5953, 1987) disclose a t-PA variant lacking carbohydrate at position 451. Van Zonneveld et al. (Proc. Natl. Acad. Sci. USA 83:4670-4674, 1986) disclose modified forms of t-PA wherein portions of the heavy chain have been deleted. Robinson et al. (EP 207,589 Al) disclose mutant forms of t-PA in which the growth factor domain has been deleted or otherwise modified. Larsen et al. (WO 87/04722) disclose t-PA-like proteins with amino acid substitutions and/or deletions. Ehrlich et al. (Fibrinolysis 1:75, 1987) disclose a recombinant t-PA molecule lacking the kringle domains. However, these variant forms of t-PA do not fully overcome the problems associated with the native protein.
There remains a need in the art for a plasminogen-activating protein with a longer half-life and specificity for fibrin. The present invention fulfills this need by providing novel derivatives of tissue plasminogen activator in which the growth factor domain has been structurally altered. The t-PA analogs described herein provide significant advantages over native t-PA as therapeutic fibrinolytic agents by permitting the use of much smaller doses, thus overcoming the problems of low solubility of native t-PA and potentially permitting administration by injection rather than infusion. Through the use of recombinant DNA technology, a consistent and homogeneous source of these proteins is provided. The proteins can be utilized to lyse existing clots in heart attack and stroke victims and in others where the need to lyse or suppress the formation of fibrin matrices is therapeutically desirable.